pSPORT-1 Library Method Overview

The laboratory of Dr. Minoru Ko constructed long-transcript enriched cDNA libraries
in the pSPORT1 vector. The method involves a PCR amplification step using
a 5' linker, LL-Sal4, and 3' primer, Sal4-S. Briefly, the method is as follows:

First strand cDNA is synthesized from total RNA using Invitrogen's SuperScriptII and
an oligo-dT primer with a NotI site [Invitrogen: 5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT-3'].

Second strand cDNA is synthesized using DNA polymerase I, and blunt ended using T4 DNA polymerase.

Purified cDNA is ligated to linker LL-Sal4 and then amplified by long-range high fidelity
PCR using Ex Taq polymerase (Takara) and primer Sal4-S.

The cDNAs are digested with SalI and NotI and cloned into the pSport1 vector.

The average insert size in different libraries ranges from 2.4 to 3.6kb.